(Circulation. 1998;98:2744-2752.)
© 1998 American Heart Association, Inc.
Basic Science Reports |
Expression of Hirudin Fusion Proteins in Mammalian Cells
A Strategy for Prevention of Intravascular Thrombosis
From the Xenotransplantation Research Group, Department of Immunology (K.R., D.C., A.J.T.G., A.D., R.I.L.) and MRC Clinical Sciences Centre Haemostasis Research Group (G.K.-C., J.H.M., E.G.D.T.), Imperial College of Science Technology and Medicine, Hammersmith Hospital, London, UK. Dr Riesbeck is presently at the Department of Medical Microbiology, University Hospital, Lund University, Malmo, Sweden.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background—Intravascular thrombosis occurs in disorders of diverse pathogeneses, including allograft and xenograft rejection. In this in vitro study, we describe an approach for tethering the specific thrombin inhibitor hirudin to plasma membranes as part of a genetic strategy for regulating intravascular coagulation.
Methods and Results—An HLA class I leader sequence was fused with hirudin linked to domains 3 and 4 of human CD4 and intracytoplasmic sequence from either CD4 or human P-selectin. The constructs were transfected into mouse fibroblasts, Chinese hamster ovary (CHO)-K1 cells, immortalized porcine endothelial cells (IPECs), and a pituitary secretory cell line (D16/16). Thrombin binding to the hirudin fusion proteins expressed on fibroblasts and CHO-K1 cells could be blocked by an anti-hirudin monoclonal antibody and by pretreatment of thrombin with either the synthetic tripeptide thrombin inhibitor PPACK or native hirudin. Hirudin expression significantly modified the procoagulant phenotype of IPECs in human plasma, leading to prolongation of clotting times. Hirudin-CD4–P-selectin fusion proteins accumulated in adrenocorticotropic hormone–containing granules in D16/16 cells, with no cell surface expression except on activation with phorbol ester, when hirudin relocated to the outer membrane.
Conclusions—Hirudin fusion proteins were expressed on mammalian cells, where they reduced local thrombin levels and inhibited fibrin formation. Regulated expression was achieved on activated cells by use of the cytoplasmic sequence from P-selectin. In vivo, these fusion proteins may prove useful transgenic or gene therapy agents for preventing intravascular thrombosis.
Key Words: anticoagulants • coagulation • transplantation • thrombosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Thrombotic vascular occlusion can occur in several multisystem disorders, may complicate local procedures such as angioplasty and vascular surgery, and features prominently in the pathology of hyperacute rejection of allografts or xenografts, where it leads to catastrophic and irreversible infarction of the transplanted organ. In this setting, the procoagulant state within the graft develops from the simultaneous effects of complement activation (dependent on the deposition of anti-graft endothelial cell [EC] antibody), subendothelial tissue factor exposure, loss of proteoglycan-linked anticoagulant molecules such as antithrombin III and tissue factor pathway inhibitor (TFPI) from EC surfaces,1 and downregulation of thrombomodulin, which is internalized after EC activation. In xenografts, another factor is the failure of some xenogeneic anticoagulant molecules to function efficiently across a species divide.2 3
Intraluminal expression of EC-tethered regulators of coagulation is a particularly attractive strategy in the context of xenotransplantation because widespread intravascular deposition of fibrin is not only the end point of hyperacute rejection but is also a prominent feature of delayed xenograft rejection.4 5 6 Other situations in which membrane-anchored anticoagulant proteins might have a therapeutic use include prevention of complications after angioplasty or vascular surgery, when transient expression within a single vessel might be a safer and more effective way to inhibit thrombosis than systemic anticoagulation.
We have previously described an engineered membrane-tethered anticoagulant protein based on the soluble plasma protein TFPI.7 In this report, we describe genetic constructs encoding other fusion proteins based on the leech anticoagulant hirudin. When expressed constitutively on the surface of mammalian cells, anchored by a portion of human CD4, hirudin retained potent thrombin-binding activity and prevented fibrin generation in vitro as determined by a plasma recalcification assay. To avoid the potential complications that would accompany constitutive anticoagulation, the hirudin-CD4 constructs were modified by the addition of P-selectin sequence to the cytoplasmic tail. This modified construct was efficiently targeted to intracellular secretory granules and was only released onto the cell surface after cellular activation. We envision these constructs will have therapeutic potential as agents for gene therapy or as transgenes for expression in donor animals bred especially for xenotransplantation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Antibodies and Reagents
The following antibodies were used in this study: mouse anti-hirudin and mouse anti–hirudin-thrombin complex (courtesy of Dr J.M. Schlaeppi, CIBA-Geigy Ltd, Basel, Switzerland); OKT-4 (ATCC); rabbit anti–human adrenocorticotropic hormone (ACTH) (Department of Histochemistry, Hammersmith Hospital, London, UK); mouse anti–human TFPI (American Diagnostica through Alpha Laboratories); rabbit anti–human prothrombin immunoglobulin (Dakopatts); mouse anti–human P-selectin (Becton Dickinson); FITC-conjugated swine anti-rabbit immunoglobulins and sheep anti-mouse immunoglobulins (Dakopatts); and mouse IgG1 and mouse IgG2a controls (Dakopatts). Native full-length hirudin was supplied by Biopharm. D-Phe-Pro-Arg chloromethyl ketone dihydrochloride (PPACK) was from Calbiochem, the chromogenic substrate H-D-Phe-Pip-Arg-pNA · 2HCl was from Quadratech, and a synthetic COOH-terminal hirudin dodecapeptide analogue (Hir53'-64' with sulfato-Tyr64') was from American Diagnostica. Other reagents were supplied by Sigma Chemical Co.
cDNA Constructs and Expression Vectors
The leader sequence of HLA-A2.1, encoding amino acids -24 to
-1, was fused to hirudin by polymerase chain reaction with
overlapping extension. The same technique was used to introduce
sequences encoding glycine-serine–rich linkers (G1 to G3) onto the 3'
end of this fusion. To link to domains 3 and 4 of human CD4
(CD4166–435), a cassette cloning strategy was
used. The P-selectin cytoplasmic region
(P-sel754–789) was introduced into some
constructs by polymerase chain reaction with overlapping extension,
replacing CD4395–435. All primers used in the
present study were designed from published cDNA sequences, and
primer sequences are available on request from the authors.
Intermediate constructs were subcloned into pBluescript SK(+)
(Stratagene) for DNA sequencing. All constructs were predicted to
encode peptides with wild-type sequence, except for the construct
encoding CD4166–435, in which
V328 was predicted to be mutated to
A328. Complete HLA-hirudin-G1 to
G3-CD4166–435 and
HLA-hirudin-G1/G2-CD4166–395-P-sel754–789
constructs were cloned into the expression vector
pHßApr-1gpt.8 Full-length human P-selectin cDNA
was subcloned into the sister vector, pHßApr-1neo. In all cases, the
orientation of ligation was determined by restriction endonuclease
mapping.
Cells, Transfections, and Flow Cytometric Analysis of
Expression Levels
The mouse fibroblast line DAP.3, mouse pituitary cell line
D16/16, and immortalized porcine endothelial cell
(IPEC) clone A89 were all grown in DMEM (Gibco).
Chinese hamster ovary (CHO)-K1 cells were maintained in RPMI 1640
(Gibco). Culture media were supplemented with 5% to 10% FCS, 10
µg/mL gentamycin, and L-glutamine. Cells were transfected
with either calcium phosphate according to standard
protocols,10 electroporation, or a modified
targeted-liposome technique. All stable transfectants were grown in
medium including mycophenolic acid (final concentration, 12
µg/mL), xanthine, and hypoxanthine or G418 as appropriate. For
staining, a standard protocol was used with 2.5% FCS in PBS as buffer.
Cells (105) were analyzed in an EPICS
XL-MCL flow cytometer (Coulter).
Immunohistochemistry
Methanol-fixed D16/16 cells were permeabilized
with 0.1% saponin and, after blocking with appropriate reagents,
incubated with rabbit anti-human ACTH immunoglobulins and a mouse
anti-hirudin monoclonal antibody (mAb) overnight. Cells were washed
twice and incubated with Texas Red–conjugated anti-rabbit IgG (Vector
Laboratories) or FITC-conjugated anti-mouse IgG detecting ACTH and
hirudin, respectively. After 30 minutes and additional washes, slides
were examined with a fluorescence microscope (Olympus Bx60;
Tokyo, Japan). Mouse anti-human TFPI mAb was used as a negative
control antibody.
Flow Cytometric Analysis of Thrombin Binding
Stably transfected cells were incubated with increasing
concentrations of thrombin at 37°C for 1 hour, then with rabbit
anti-human prothrombin immunoglobulins for 30 minutes on ice, followed
finally by FITC-conjugated swine anti-rabbit immunoglobulins. In
blocking experiments, cells were first incubated with anti-hirudin
antibodies or appropriate control immunoglobulins for 30 minutes on ice
and washed before being incubated with thrombin for 1 hour at 37°C as
described above.
Inactivation of Thrombin Assessed by Amidolytic Assay
Either PPACK11 or a dodecapeptide
corresponding to the C-terminal sequence of hirudin was incubated at
100-fold molar excess with 210 nmol of thrombin in 50 µL of
Tris-buffered saline, 0.1% BSA, pH 7.4 for 1 hour at 37°C. In
parallel, thrombin was treated with a 10-fold molar excess of native
hirudin for 1 hour at 37°C. Thrombin activity was assessed by
absorbance at 405 nm in a Thermomax microplate reader (Molecular
Devices Ltd) after incubation with the substrate S-2238 for 10 minutes
at 37°C.
Modulation of EC Procoagulant Phenotype in Human Plasma by
Hirudin Expression
We mixed 107 hirudin-CD4–transfected
IPECs or control untransfected IPECs with normal human plasma (Sigma)
and 12 mmol/L CaCl2 in glass tubes (Corning
Inc). The time for a fibrin clot to form at 37°C was determined in
triplicate by a standard tilting technique. In some experiments, IPECs
were first preincubated for 10 hours with human recombinant
interleukin-1
(IL-1) (10 ng/mL; Boehringer Mannheim) to
induce tissue factor expression before washing and inclusion in
clotting assays.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Hirudin Fused With HLA Class I Signal Peptide and Linked to Domains 3 and 4 of Human CD4 Is Expressed as a Transmembrane Protein
The different hirudin constructs used in the present study are shown in Figure 1
. Three
different glycine-rich linkers (designated G1 to G3) were used (Figure 1A) to minimize the risk of steric interference of the interaction
between hirudin and thrombin by the immunoglobulin-like third domain of
CD4.
|
On stable DAP.3 transfectants, hirudin-CD4 was easily detected at the
cell surface (Figure 2), and its
expression appeared uninfluenced by the length of the glycine linker.
If trypsin was used to harvest cells from culture, the hirudin moiety
was cleaved off whereas the CD4 anchor was left intact (Figure 2G and 2H), indicating that a trypsin cleavage recognition sequence may exist
between hirudin and CD4.
|
Hirudin Anchored to the Cell Surface Binds Thrombin
After incubation with thrombin at 37°C, only
hirudin-CD4–expressing DAP.3 cells and not untransfected controls
bound thrombin in a dose-dependent manner (Figure 3A and 3B); the type of glycine linker
had no influence on thrombin binding. However, binding was inhibited if
transfectants were preincubated with an anti-hirudin mAb (Figure 3C).
Thrombin binding was also detected with an mAb directed against the
hirudin-thrombin complex (Figure 3D). Because this antibody recognizes
neither hirudin in the absence of thrombin nor thrombin complexed with
endogenous thrombin receptors,12
these observations, taken together, demonstrate specific thrombin
binding by the surface-expressed hirudin-CD4 fusion protein.
|
), HLA-hirudin-G2-CD4 (
),
HLA-hirudin-G3-CD4 (
), or the negative control HLA-DR (
) were
incubated with thrombin for 1 hour at 37°C. Percentage positive cells
(A) and mean fluorescence intensities (B) are shown. Cells
were labeled with an anti-prothrombin immunoglobulin fraction.
Representative of 2 analyses. C, DAP.3 cells
transfected with HLA-hirudin-G3-CD4 were incubated with either 10
µg/mL anti-hirudin mAb (
) or a specific
anti-hirudin-thrombin mAb (
). Representative of 3
analyses. E, Active-site blocking of thrombin abolishes
thrombin binding to hirudin-CD4 at the cell surface. DAP.3 cells
expressing hirudin-G2-CD4 were incubated with thrombin or thrombin
pretreated with various inhibitors and subsequently stained
for thrombin binding with anti-prothrombin immunoglobulins or
anti-thrombin-hirudin mAb according to the standard procedure (see
Methods and Results). Representative of 2
experiments.
Interaction Between Thrombin and Hirudin-CD4 Is Abolished When the
Catalytic Site of Thrombin Is Blocked
The amino-terminal domain of native hirudin binds to the active
(catalytic) site of thrombin, whereas the carboxy-terminal binds to the
separate anion-binding exosite of thrombin.13
Thrombin binding to the hirudin-CD4 fusion protein was assessed after
preincubation of the enzyme with either native hirudin, the tripeptide
active site inhibitor PPACK, or a synthetic COOH-terminal
hirudin dodecapeptide. Before analysis of binding, residual
thrombin-dependent catalytic activity was analyzed with a small
oligopeptide substrate (S-2238). As expected, thrombin preincubated
with dodecapeptide cleaved S-2238 normally, whereas both native hirudin
and PPACK completely abolished thrombin amidolytic activity (data not
shown). As can be seen in Figure 3E
, thrombin pretreated with hirudin
or PPACK was not bound by cell-surface hirudin-CD4, whereas treatment
with the hirudin dodecapeptide only slightly reduced thrombin binding.
In conclusion, these results indicate that hirudin-CD4 tethered to the
cell surface specifically and strongly bound thrombin via its catalytic
site.
Hirudin-CD4 Expressed by IPECs Binds Thrombin and Inhibits
Coagulation in Human Plasma
Figure 4A and 4B demonstrates that
when expressed on IPECs, hirudin-CD4 bound thrombin similarly as when
expressed on transfected DAP.3 cells.
|
In a simple clotting assay, untransfected IPECs shortened the clotting
time of citrated human plasma after recalcification to 
170 seconds,
compared with a control clotting time of 370 seconds in the absence of
cells (Figure 4C). After preincubation of untransfected IPECs with
IL-1, clotting times were further reduced to <100 seconds owing to
the expression of tissue factor (as confirmed by flow cytometric
analysis; data not shown). In contrast, the clotting times
recorded with IPECs expressing hirudin-CD4 were
consistently prolonged compared with those with untransfected
control cells, even after preincubation in IL-1 and induction of
tissue factor expression. This effect was due to the presence of cell
surface–expressed hirudin on the transfected IPECs, as demonstrated by
the attenuation of the prolongation of clotting by anti-hirudin mAb
(Figure 4D).
Hirudin-CD4 With a Targeting Sequence From the COOH-Terminal Domain
of P-Selectin Is Functionally Similar to Hirudin-CD4
Two constructs, with a targeting sequence derived
from human P-selectin but with different glycine linkers, were
synthesized (Figure 1B) and used to establish stable CHO-K1
transfectants. These cells were chosen because they lack secretory
granules. In control experiments, cells transfected with wild-type
P-selectin expressed protein at the cell surface (data not shown),
which confirms what others have documented.14
Consistent with this, both hirudin-CD4–P-selectin constructs
were expressed at the cell surface of the CHO-K1 transfectants (data
not shown). Once again, the length of the glycine linker had no
influence on surface expression. Thrombin binding to these constructs
was investigated in the same manner as for the hirudin-CD4–transfected
DAP.3 clones and found to be identical in all respects (data not
shown). We concluded from these results that inclusion of the
P-selectin cytoplasmic tail had no adverse influence on the
thrombin-binding activity of hirudin.
Hirudin-CD4–P-Selectin Is Stored in Secretory Granules in
Pituitary Cells and Can Be Released on Activation
To examine whether hirudin-CD4–P-selectin accumulated in
intracellular storage granules, we transiently transfected a secretory
mouse pituitary cell line (D16/16) with either hirudin-CD4–P-selectin
or hirudin-CD4. This particular cell line was used for 2 reasons.
First, the cells have been previously described to express ACTH in
specific storage granules that are discharged to the cell surface on
activation with phorbol esters. Second, pig aortic ECs do not contain
Weibel-Palade (W-P) storage granules,15 and other
vascular ECs rapidly lose their W-P bodies on culture in
vitro.
In D16/16 cells transfected with hirudin-CD4–P-selectin, hirudin was
detected in granules evenly distributed in the cytoplasm (Figure 5A; green fluorescence). The same
pattern of granule distribution was detected when cells were examined
for ACTH expression, which implies colocalization with hirudin (Figure 5B; red fluorescence). This finding was verified and was even
more striking in specimens examined for both hirudin and ACTH (yellow
granules, indicating colocalization, Figure 5C). Moreover, D16/16 cells
that were transfected with hirudin-CD4 cDNA (lacking the P-selectin
sequence) did not accumulate hirudin in intracellular granules but
expressed high levels of hirudin at the cell surface (Figure 5D). When
these transfectants were examined for both ACTH and hirudin staining,
no colocalization was observed (orange granules, Figure 5F), which
contrasts markedly with the cells transfected with
hirudin-CD4–P-selectin (Figure 5C).
|
In support of this interpretation, D16/16 cells stably transfected with
hirudin-CD4–P-selectin did not express detectable hirudin at the
surface after being stained with anti-hirudin mAb (Figure 6A). However, after stimulation with
phorbol myristate acetate (PMA) for 30 minutes, hirudin
was relocated to the cell surface (Figure 6B). Furthermore,
PMA-activated D16/16 cells specifically bound thrombin, as
shown with the anti-hirudin-thrombin–specific mAb (Figure 6C), unlike
the nonactivated cells (not shown). Thus, using the
granule-containing pituitary cell line D16/16, we clearly demonstrated
that hirudin-CD4–P-selectin could be targeted to specific storage
granules and that functional fusion proteins were released and exposed
at the cell surface on cellular activation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, using novel fusion proteins, we have demonstrated both constitutive expression of cell surface–tethered hirudin and stimulation-dependent relocation of hirudin from secretory granules to the cell surface. Linked to an appropriate mammalian signal sequence and membrane anchor, hirudin was expressed at the cell surface, where it bound exogenous thrombin via its active site and inhibited the formation of fibrin in an in vitro clotting assay that used human plasma. When the C-terminal sequence of the hirudin-CD4 fusion protein was replaced by a targeting sequence derived from P-selectin, it accumulated in storage granules of secretory cells and could be relocated to the cell surface on activation. Furthermore, this relocated hirudin was active, because specific thrombin binding could be visualized by flow cytometry.
The targeting and regulated expression of the P-selectin–tagged fusion protein were predictable from recent data concerning the intracellular processing of P-selectin.16 17 18 Indeed, previous work has shown that fusion of the cytoplasmic sequence of P-selectin can redirect protein expression into storage granules.14 We have shown for the first time that expression of hirudin can also be manipulated by targeting to storage granules as part of a novel genetic strategy to tether functionally active anticoagulant molecules to EC surfaces.
The efficacy of recombinant hirudin in various preclinical and clinical models of thrombosis is well documented.19 20 21 22 At least 1 soluble fusion protein containing hirudin has been shown to retain thrombin binding activity in vitro.23 Hirudin has also been successfully targeted to specific sites by use of covalently linked antibodies or Fab fragments to fibrin24 and E-selectin (CD62E), which is expressed on activated ECs.25 More recently, adenovirus-mediated gene transfer leading to expression of recombinant soluble hirudin in smooth muscle cells of injured rat carotid arteries was shown to be effective in the inhibition of neointima formation,26 which suggests that local delivery of hirudin may constitute an attractive intervention strategy for arterial proliferative disease.
These approaches may, however, be problematic in that they rely on treating thrombosis that is already well established, whereas our genetic approach is designed to inhibit trace thrombin generation before clot formation. We envision that the hirudin-CD4 constructs could be used proactively in situations in which thrombotic complications are anticipated, including vascular surgery and transplantation. After therapy with the hirudin-CD4–P-selectin constructs, constitutive hirudin expression would be absent on normal, quiescent endothelium but would be rapidly expressed on type 1 EC activation induced by complement activation or small amounts of thrombin, owing to the discharge of W-P bodies. This strategy should have clear advantages over other targeted anticoagulant strategies that rely on constitutive expression.
In transplantation, EC activation and P-selectin expression occur in allografts subject to prolonged hypoxia and hypothermic preservation, and these grafts are susceptible to reperfusion injury.27 Similar changes occur during hyperacute rejection and delayed xenograft rejection.6 In these settings, expression of hirudin-CD4–P-selectin fusion is expected to have beneficial effects on graft survival by inhibiting intraluminal fibrin deposition. In combination with complement inhibitory proteins at the EC surface, the expression of hirudin activity in xenografts may be a potent strategy in prevention of the microvascular thrombosis associated with all types of vascular rejection.
|
Acknowledgments |
|---|
This study was funded by grants from the Medical Research Council and PPL Therapeutics. Special thanks to Dr P. Sarmientos (Prodotti Immunobiotecnologici, Milan, Italy) for hirudin variant 1 cDNA, Dr A. Madrigal (Royal Free Hospital, London, UK) for HLA A2.1 cDNA, and Dr R.P. McEver (Department of Biochemistry, University of Oklahoma, Oklahoma City) for human P-selectin cDNA.
|
Footnotes |
|---|
Reprint requests to Robert Lechler, Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Rd, London W12 0NN, UK.
Received March 27, 1998; revision received August 7, 1998; accepted August 19, 1998.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
-
Platt JL, Vercellotti GM, Lindman BJ, Oegema TR,
Bach FH, Dalmasso AP. Release of heparan sulphate from
endothelial cells: implications for pathogenesis of
hyperacute rejection. J Exp Med. 1990;171:1363–1368.
[Abstract/Free Full Text]
-
Kopp CW, Siegel JB, Hancock WW, Anrather J, Winkler H,
Geczy CL, Kaczmarek E, Bach FH, Robson SC. Effect of porcine
endothelial tissue factor pathway inhibitor
on human coagulation factors. Transplantation. 1997;63:749–758.[Medline]
[Order article via Infotrieve]
-
Siegel JB, Grey ST, Lesnikoski BA, Kopp CW, Soares M,
Schulte am Esch J II, Bach FH, Robson SC. Xenogeneic
endothelial cells activate human prothrombin.
Transplantation. 1997;64:888–896.[Medline]
[Order article via Infotrieve]
-
McCurry KR, Kooyman DL, Alvarado CG, Cotterell
AH, Martin MJ, Logan JS, Platt JL. Human complement regulatory proteins
protect swine-to-primate cardiac xenografts from humoral injury.
Nat Med. 1995;1:423–427.[Medline]
[Order article via Infotrieve]
-
Rosengard AM, Cary NRB, Langford GA, Tucker AW,
Wallwork J, White DJG. Tissue expression of human complement
inhibitor, decay-accelerating factor, in transgenic pigs.
Transplantation. 1995;59:1325–1333.[Medline]
[Order article via Infotrieve]
-
Bach FH, Winkler H, Ferran C, Hancock WW, Robson SC.
Delayed xenograft rejection. Immunol Today. 1996;17:379–384.[Medline]
[Order article via Infotrieve]
-
Riesbeck K, Dorling A, Kemball-Cook G, McVey JH, Jones
M, Tuddenham EG, Lechler RI. Human tissue factor pathway
inhibitor fused to CD4 binds both FXa and TF/FVIIa at the
cell surface. Thromb Haemost. 1997;78:1488–1494.[Medline]
[Order article via Infotrieve]
-
Gunning P, Leavitt J, Muscat G, Ng SY, Kedes L. A
human ß actin expression vector system directs high level
accumulation of anti-sense transcripts. Proc Natl Acad Sci
U S A. 1987;84:4831–4835.
[Abstract/Free Full Text]
-
Dorling A, Stocker C, Tsao T, Haskard DO, Lechler RI.
In vitro accommodation of immortalized porcine
endothelial cells: resistance to complement mediated
lysis and down-regulation of VCAM expression induced by low
concentrations of polyclonal human IgG antipig antibodies.
Transplantation. 1996;62:1127–1136.[Medline]
[Order article via Infotrieve]
-
Margulies DH, Evans GA, Ozato K, Camerini OR, Tanaka K,
Appella E, Seidman JG. Expression of H-2Dd and H-2Ld mouse major
histocompatibility antigen genes in L cells after DNA-mediated gene
transfer. J Immunol. 1983;130:463–470.[Abstract]
-
Kettner C, Shaw E. D-Phe-Pro-ArgCH2C1: a selective
affinity label for thrombin. Thromb Res. 1979;14:969–973.[Medline]
[Order article via Infotrieve]
-
Schlaeppi JM. Preparation of monoclonal antibodies to
the thrombin/hirudin complex. Thromb Res. 1991;62:459–470.[Medline]
[Order article via Infotrieve]
-
Stone SR, Betz A, Parry MA, Jackman MP, Hofsteenge J.
Molecular basis for the inhibition of thrombin by hirudin. Adv
Exp Med Biol. 1993;340:35–49.[Medline]
[Order article via Infotrieve]
-
Disdier M, Morrissey JH, Fugate RD, Bainton DF, McEver
RP. Cytoplasmic domain of P-selectin (CD62) contains the signal for
sorting into the regulated secretory pathway. Mol Biol Cell. 1992;3:309–321.[Abstract]
-
Rand JH, Badimon L, Gordon RE, Uson RR, Fuster V.
Distribution of von Willebrand factor in porcine intima varies
with blood vessel type and location.
Arteriosclerosis. 1987;7:287–291.
[Abstract/Free Full Text]
-
Wagner DD. The Weibel-Palade body: the storage granule
for von Willebrand factor and P-selectin. Thromb
Haemost. 1993;70:1053–1057.[Medline]
[Order article via Infotrieve]
-
Diacovo TG, Roth SJ, Morita CT, Rosat JP, Brenner
MB, Springer TA. Interactions of human alpha/beta and gamma/delta T
lymphocyte subsets in shear flow with E-selectin and P-selectin.
J Exp Med. 1996;183:1193–1203.
[Abstract/Free Full Text]
-
Austrup F, Vestweber D, Borges E, Lohning M, Brauer R,
Herz U, Renz H, Hallmann R, Scheffold A, Radbruch A, Hamann A. P- and
E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells
into inflamed tissues. Nature. 1997;385:81–83.[Medline]
[Order article via Infotrieve]
-
Kirchhofer D, Tschopp TB, Hadvary P, Baumgartner HR.
Endothelial cells stimulated with tumor necrosis
factor-alpha express varying amounts of tissue factor resulting in
inhomogeneous fibrin deposition in a native blood flow
system: effects of thrombin inhibitors. J Clin
Invest. 1994;93:2073–2083.
-
A comparison of recombinant hirudin with heparin for
the treatment of acute coronary syndromes: the Global Use of
Strategies to Open Occluded Coronary Arteries (GUSTO) IIb
investigators. N Engl J Med. 1996;335:775–782.
[Abstract/Free Full Text]
-
Eriksson BI, Ekman S, Kalebo P, Zachrisson B, Bach D,
Close P. Prevention of deep-vein thrombosis after total hip
replacement: direct thrombin inhibition with recombinant hirudin, CGP
39393. Lancet. 1996;347:635–639.[Medline]
[Order article via Infotrieve]
-
Mruk JS, Zoldhelyi P, Webster MW, Heras M, Grill DE,
Holmes DJ, Fuster V, Chesebro JH. Does antithrombotic therapy influence
residual thrombus after thrombolysis of
platelet-rich thrombus? Effects of recombinant hirudin, heparin, or
aspirin. Circulation. 1996;93:792–799.
[Abstract/Free Full Text]
-
Lijnen HR, Wnendt S, Schneider J, Janocha E, Van HB,
Collen D, Steffens GJ. Functional properties of a recombinant chimeric
protein with combined thrombin inhibitory and
plasminogen-activating potential. Eur J
Biochem. 1995;234:350–357.[Medline]
[Order article via Infotrieve]
-
Bode C, Hudelmayer M, Mehwald P, Bauer S, Freitag M,
von HE, Newell JB, Kubler W, Haber E, Runge MS. Fibrin-targeted
recombinant hirudin inhibits fibrin deposition on experimental clots
more efficiently than recombinant hirudin. Circulation. 1994;90:1956–1963.
[Abstract/Free Full Text]
-
Kiely JM, Cybulsky MI, Luscinskas FW, Gimbrone MJ.
Immuno-selective targeting of an anti-thrombin agent to the surface
of cytokine-activated vascular
endothelial cells. Arterioscler Thromb Vasc
Biol. 1995;15:1211–1218.
[Abstract/Free Full Text]
-
Rade JJ, Schulick AH, Virmani R, Dichek DA. Local
adenoviral-mediated expression of recombinant hirudin reduces
neointima formation after arterial injury.
Nat Med. 1996;2:293–298.[Medline]
[Order article via Infotrieve]
-
Pinsky DJ, Oz MC, Koga S, Taha Z, Broekman MJ,
Marcus AJ, Liao H, Naka Y, Brett J, Cannon PJ, Nowygrod R, Malinski T,
Stern D. Cardiac preservation is enhanced in a heterotopic rat
transplant model by supplementing the nitric oxide pathway.
J Clin Invest. 1994;93:2291–2297.
This article has been cited by other articles:
 |
|
 |
|
  D. Chen, K. Giannopoulos, P. G. Shiels, Z. Webster, J. H. McVey, G. Kemball-Cook, E. Tuddenham, M. Moore, R. Lechler, and A. Dorling Inhibition of intravascular thrombosis in murine endotoxemia by targeted expression of hirudin and tissue factor pathway inhibitor analogs to activated endothelium Blood, September 1, 2004; 104(5): 1344 - 1349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M D Dooldeniya and A N Warrens Xenotransplantation: where are we today? J R Soc Med, March 1, 2003; 96(3): 111 - 117. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||